Whole virus vaccines against HIV, live attenuated or killed offer the potential to stimulate immunity to the broadest array of antigenic determinants of the virus. However, they also may contain structures developed by the virus to evade the immune system, such as suppressive epitopes or masking carbohydrates, or structures which elicit deleterious effects such as enhancing antibodies that increase viral infectivity (Takeda, A. et al. Science 242:580-583. (1988); Robinson, W. E. Jr. et al., Proc. Nati. Acad. Sci. USA 86:4710-4714 (1989); Robinson, W. E., Jr. et al.; Proc. Natl. Acad. Sci. USA 87:3185-3189 (1990); Halstead, S. B. Science 239:476-481 (1988)) or antibodies or T cells that may contribute to immunodeficiency in the case of HIV (Weinhold, K. J. et al., J. Immununol 142:3091-3097 (1989); Siliciano, R-F.; et al., Cell 54:561-575 (1988); Mittler, R. S. and M. K. Hoffmann, Science 245:1380-1382 (1989); Golding, H. et al., J. Clin. Invest. 83:1430-1435 (1989)). In addition, for a retrovirus such as HIV, concerns about the safety of live attenuated or even killed whole viral vaccines may make them unacceptable to many potential recipients. Purified subunit vaccines have less safety risk, but still may suffer from the other problems of whole virus vaccines. Indeed, because the virus has evolved to evade the immune system, evolution may have favored the development of viral proteins that are hardly optimal as vaccines. Thus, in contrast to enzymes which have been honed by evolution to be the best structures for catalyzing their reactions, viral proteins may leave the scientist with considerable opportunities to improve on nature for the development of better vaccines (Berzofsky, J. A., J. Clin. Invest. 82:1811-1817 (1988)).
To rationally design highly engineered synthetic or recombinant antiviral vaccines, one needs considerable knowledge about the workings of the immune system, and in particular, about the immune response to structures expressed by the virus. The present inventors have initiated such an approach by attempting to identify antigenic determinants recognized by cytotoxic T lymphocytes (CTL) (Takahashi, H. et al., Proc. Natl. Acad. Sci. USA 85:3105-3109 (1988); Takahashi, H. et al, Science 246:118-121 (1989); Takahashi, H. et al., J. Exp. Med. 170:2023-2035 (1989); Takahashi, H. et al., J. Exp. Med. 171:571-576 (1990); Hosmalin, A. et al, Proc. Natl. Acad. Sci. USA 87:2344-2348 (1990)) and by helper T cells that would be required for either a CTL or an antibody response (Cease, K. B. et al., Proc. Natl. Acad. Sci. USA 84:4249-4253 (1987); Berzofsky, J. A. et al. Nature 334:706-708 (1988); Clerici, M. et al., Nature 339:383-385 (1989); Hale, P. M. et al., Internat. Immunol. 1:409-415 (1989)). However, a potential problem with the use of any single antigenic determinant is that T cells recognize antigens in association with molecules encoded by the major histocompatibility complex (MHC) of the host, and the MHC molecules of any given individual will bind and present only a subset of potential antigenic determinants that could be recognized by the species as a whole (Benacerraf, B., J. Immunol. 120:1809-1812 (1978); Schwartz, R. H., Annu. Rev. Immunol. 3:237-261 (1985); Berzofsky, J. A., in "The Antigens". pp. 1-146, M. Sela, editor, c. 1987 by Academic Press, New York). This is true of humans as well as mice (Siliciano, R-F. et al, Cell 54:561-575 (1988); Schrier, R. D. et al., J. Immunol. 142:1166-1176 (1989); Callahan, K. M. et al, J. Immunol. 144:3341-3346 (1990); Martin, R. et al., J. Immunol. 145:540-548 (1990); Martin, R. et al., J. Exp. Ned. 173:19-24 (1991); Jaraquemada, D. et al., J. Immunol. 145:2880-2885 (1990)).
Therefore, in order to be useful in a broad outbred population such as humans, a vaccine should contain multiple such determinants. Only limited data exist to indicate how many such determinants would have to be included. Although some concern has been raised that the number might be impractical to achieve, some data exist to suggest that as few as four such determinants could elicit responses in 85-90% of outbred humans (Clerici, M. et al., Nature 339:383-385 (1989)). A few antigenic peptides have been identified that appear to be promiscuous in their recognition in association with many DR molecules (sinigaglia, F. et al., Nature 336:778-780 (1988); Panina-Bordignon, P. et al., Eur. J. Immunol. 19:2237-2242 (1989)), perhaps because DR molecules share a conserved alpha chain, and in the mouse some determinants have been reported to be presented by three different I-A molecules that do not share alpha chains (Brett, S. J. et al., J. Immunol. 143:771-779 (1989)), or even by class II MHC molecules of different isotypes, such as I-A and I-E (Guillet, J.-G. et al., Science 235:865-870 (1987))). However, it is not clear how common such promiscuous epitopes are.
In the course of locating the major T-cell stimulatory sites of the HIV envelope, we observed that there were regions in the sequence that contained multiple overlapping determinants seen by mice of different MHC haplotypes (Hale, P. M. et al., Internat. Immunol. 1:409-415 (1989)). Although the precise determinants seen by T cells of each strain of mouse differed, each multideterminant region contained determinants that could stimulate T cells of mice of three or four of the four MHC types tested. We, therefore, reasoned that peptides encompassing such multideterminant regions might be able to stimulate T cells of many or most haplotypes of mice, and hopefully also T cells of humans of many HLA types. Thus, such multideterminant peptides might provide a means to circumvent this problem of MHC restriction in the design of synthetic vaccines. The present applicants have, therefore, tested this hypothesis by constructing six synthetic peptides of 20-33 residues each that correspond to the six multideterminant regions of HIV envelope protein localized in the mouse (Hale, P. M. et al., Internat. Immunol. 1:409-415 (1989)), and tested these peptides for their ability to stimulate T-cell responses in mice immunized with recombinant gp160 and in peripheral blood lymphocytes of humans infected with HIV. Although not all of the peptides were as widely recognized as expected, several such peptides were identified that were broadly recognized by both murine and human T cells of multiple H-2 and HIA types. These peptides can also immunize mice for T-cell responses to the intact HIV envelope protein, and so are useful as valuable components of a synthetic vaccine, and responses to them are useful diagnostic or prognostic markers.